Poster: Membrane transport

Abs # 167: Expression of Arabidopsis cyclic nucleotide gated nonselective cation channels (CNGCs) in yeast: Problems and prospective

Presenter:	Ali, Rashid , rali@canr.uconn.edu
Authors	Ali, Rashid  (A)   Berkowitz, Gerald A (A)
Affiliations:	(A): University of Connecticut

Mutants of yeast Saccharomyces cerevisiae have been extensively used to isolate and express heterologous transport proteins. Little attention has been paid to the problem of expression, processing, and intracellular localization of these recombinant proteins in yeast. Furthermore, the fact that yeast mutants can adapt, depending on environmental and growth conditions, by activating alternate pathways, and that adaptation could have significant effects on the functional assay of the heterologous protein, have not been given due consideration. In the present study we address these issues. Under assay conditions that we have developed, expression of AtCNGC1,2 and 4 results in different responses of the K⁺ uptake deficient mutant trk1,2 in terms of growth at low K⁺, and growth in the presence of hygromycin. Inhibitors of the Na⁺/H⁺-antiporter (amiloride analogues) improve these functional assays of the AtCNGCs. AtCNGC2 was able to support growth at low K⁺, while growth complementation was less strong with AtCNGC1 and 4, although northern analyses and confocal microscopy of GFP-tagged AtCNGCs showed that the genes were expressed and localized to the plasma membrane. Surprisingly, deletion of the calmodulin binding domain (CaMBD) at the C-terminus of AtCNGC1 resulted in a functional phenotype and stronger complementation. Similar mutations of AtCNGC4 showed no effect. This suggests that AtCNGCs may be subjected to varying degrees to host regulatory mechanisms, possibly through protein-protein interactions of yeast calmodulin with the AtCNGC CaMBD. Further investigation in this regard is in progress using two-hybrid interactions to study calmodulin binding, and intracellular Na and K measurements to confirm the growth complementation phenotypes. Supported by NSF award 0344141.